Becker John V W, Armstrong Gareth O, van der Merwe Marthinus J, Lambrechts Marius G, Vivier Melané A, Pretorius Isak S
Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, 7600 Stellenbosch, South Africa.
FEMS Yeast Res. 2003 Oct;4(1):79-85. doi: 10.1016/S1567-1356(03)00157-0.
The stilbene resveratrol is a stress metabolite produced by Vitis vinifera grapevines during fungal infection, wounding or UV radiation. Resveratrol is synthesised particularly in the skins of grape berries and only trace amounts are present in the fruit flesh. Red wine contains a much higher resveratrol concentration than white wine, due to skin contact during fermentation. Apart from its antifungal characteristics, resveratrol has also been shown to have cancer chemopreventive activity and to reduce the risk of coronary heart disease. It acts as an antioxidant and anti-mutagen and has the ability to induce specific enzymes that metabolise carcinogenic substances. The objective of this pilot study was to investigate the feasibility of developing wine yeasts with the ability to produce resveratrol during fermentation in both red and white wines, thereby increasing the wholesomeness of the product. To achieve this goal, the phenylpropanoid pathway in Saccharomyces cerevisiae would have to be introduced to produce p-coumaroyl-CoA, one of the substrates required for resveratrol synthesis. The other substrate for resveratrol synthase, malonyl-CoA, is already found in yeast and is involved in de novo fatty-acid biosynthesis. We hypothesised that production of p-coumaroyl-CoA and resveratrol can be achieved by co-expressing the coenzyme-A ligase-encoding gene (4CL216) from a hybrid poplar and the grapevine resveratrol synthase gene (vst1) in laboratory strains of S. cerevisiae. This yeast has the ability to metabolise p-coumaric acid, a substance already present in grape must. This compound was therefore added to the synthetic media used for the growth of laboratory cultures. Transformants expressing both the 4CL216 and vst1 genes were obtained and tested for production of resveratrol. Following beta-glucosidase treatment of organic extracts for removal of glucose moieties that are typically bound to resveratrol, the results showed that the yeast transformants had produced the resveratrol beta-glucoside, piceid. This is the first report of the reconstruction of a biochemical pathway in a heterologous host to produce resveratrol.
芪类化合物白藜芦醇是酿酒葡萄在真菌感染、受伤或紫外线辐射期间产生的一种应激代谢产物。白藜芦醇尤其在葡萄浆果的果皮中合成，果肉中仅含有微量。由于发酵过程中与果皮接触，红酒中的白藜芦醇浓度比白酒高得多。除了具有抗真菌特性外，白藜芦醇还被证明具有癌症化学预防活性，并能降低冠心病风险。它作为一种抗氧化剂和抗诱变剂，能够诱导代谢致癌物质的特定酶。这项初步研究的目的是探讨开发具有在红葡萄酒和白葡萄酒发酵过程中产生白藜芦醇能力的葡萄酒酵母的可行性，从而提高产品的健康价值。为实现这一目标，必须在酿酒酵母中引入苯丙烷类途径以产生对香豆酰辅酶A，这是白藜芦醇合成所需的底物之一。白藜芦醇合酶的另一种底物丙二酰辅酶A已在酵母中发现，并参与脂肪酸的从头生物合成。我们假设，通过在酿酒酵母实验室菌株中共表达来自杂种杨树的辅酶A连接酶编码基因（4CL216）和葡萄白藜芦醇合酶基因（vst1），可以实现对香豆酰辅酶A和白藜芦醇的生产。这种酵母具有代谢对香豆酸的能力，对香豆酸是葡萄汁中已存在的一种物质。因此，将该化合物添加到用于实验室培养物生长的合成培养基中。获得了同时表达4CL216和vst1基因的转化体，并对白藜芦醇的生产进行了测试。在用β-葡萄糖苷酶处理有机提取物以去除通常与白藜芦醇结合的葡萄糖部分后，结果表明酵母转化体产生了白藜芦醇β-葡萄糖苷，即白藜芦醇苷。这是在异源宿主中重建生化途径以生产白藜芦醇的首次报道。
